A double-stranded RNA mycovirus in Botrytis cinerea

In wild-type Botrytis cinerea CVg25 strain we have detected the presence of extrachromosomal genetic elements corresponding to double-stranded RNA molecules. These genetic elements have been designated L, M₁ and M₂ with molecular sizes of 8.3, 2.0 and 1.4 kb, respectively. The visualization by electron microscopy of mycelium ultrathin sections from B. cinerea CVg25 showed the presence of isometric virus-like particles of about 40 nm in diameter. Linear sucrose gradient centrifugation of mycelium-free extracts was done to determine if the double-stranded RNAs were associated with virus-like particles. The gradient profile obtained at 260 and 280 nm revealed a major peak that was analyzed by both agarose-gel electrophoresis and electron microscopy. It was observed that only the L-double-stranded RNA molecule copurified with isometric virus-like particles. These virus-like particles had a similar morphology and size as those detected by electron microscopy in the mycelium sections. These results suggest that only the L-double-stranded RNA would be encapsidated.     

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic-polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-gamma, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 microM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.     

Functional comparison of single- and double-stranded siRNAs in mammalian cells

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.     

Characterization of the double-stranded RNA responses in human liver progenitor cells

Human HepaRG cells are liver progenitors which possess hepatocyte-like functionality. We investigated the effects of double-stranded (ds) RNA on interferon (IFN)-β and chemokine (CK) expression in these cells. By microarray and ELISA, we showed strong induction of CXCL10 and interleulin (IL)-8 besides IFN-β and other CK ligands. RNA interference directed silencing of TLR3, RIG-I, IRF3, NFκB or MAP kinases (p38, ERK, JNK) was carried out. Knockdown of all these molecules, except ERK and JNK, blocked IFN-β production. Both TLR3 and RIG-I are required for CXCL10 expression. Silencing of TLR3 completely impaired the IL-8 expression. dsRNA-conditioned medium from HepaRG cells exerted a drastic antiviral effect in HCV replicons, and in the JFH-1-based HCV production cell culture system. The IFN-β knockdown in HepaRG cells removed this antiviral effect but did not enhance their capacity to initiate HCV RNA replication. We conclude that dsRNA induces antiviral and pro-inflammatory status in HepaRG cells.     

Inactivation of the type I interferon pathway reveals long double‐stranded RNA‐mediated RNA interference in mammalian cells

RNA interference (RNAi) elicited by long double‐stranded (ds) or base‐paired viral RNA constitutes the major mechanism of antiviral defence in plants and invertebrates. In contrast, it is controversial whether it acts in chordates. Rather, in vertebrates, viral RNAs induce a distinct defence system known as the interferon (IFN) response. Here, we tested the possibility that the IFN response masks or inhibits antiviral RNAi in mammalian cells. Consistent with that notion, we find that sequence‐specific gene silencing can be triggered by long dsRNAs in differentiated mouse cells rendered deficient in components of the IFN pathway. This unveiled response is dependent on the canonical RNAi machinery and is lost upon treatment of IFN‐responsive cells with type I IFN. Notably, transfection with long dsRNA specifically vaccinates IFN‐deficient cells against infection with viruses bearing a homologous sequence. Thus, our data reveal that RNAi constitutes an ancient antiviral strategy conserved from plants to mammals that precedes but has not been superseded by vertebrate evolution of the IFN system.     

High-frequency occurrence of virus-like particles with double-stranded RNA genome in Fusarium poae

Abstract Fifty-five geographically different strains of Fusarium poae were assayed for the presence of extrachromosomal nucleic acid elements. All strains were found to harbour double-stranded RNA (dsRNA) elements and encapsidated virus-like particles (VLP). There were great individual differences in dsRNA patterns of the various strains, but numbers and sizes characteristic for a given isolate remained unchanged after repeated subculturing of the fungi. Morphological alterations or signs of degeneration were not observed in dsRNA-containing isolates. This is the first report on the ubiquitous occurrence of dsRNAs in a hyphomycete fungus species.     

Cationic lipids enhance siRNA-mediated interferon response in mice

RNA interference mediated by small interfering RNAs (siRNAs) shows promise as a powerful research tool for gene function studies. However, controversy exists over the potential of siRNA-induced interferon response in vitro and in vivo. In this study, we showed that although intravenous administration of siRNA alone is essentially inert, injection of siRNA complexed with cationic liposomes resulted in a potent induction of both type I and type II interferon responses. Furthermore, i.v. administration of cationic lipid/siRNA complexes led to activation of STAT1. This study suggests caution in data interpretation and the potential toxicity with in vivo use of siRNA, particularly when delivered via a cationic lipid vector. This study also suggests the potential of siRNA as an immunostimulatory agent for immunotherapy.     

Extra double-stranded RNA binding domain (dsRBD) in a squid RNA editing enzyme confers resistance to high salt environment

A-to-I RNA editing is particularly common in coding regions of squid mRNAs. Previously, we isolated a squid editing enzyme (sqADAR2) that shows a unique structural feature when compared with other ADAR2 family members: an additional double-stranded RNA (dsRNA) binding domain (dsRBD). Alternative splicing includes or excludes this motif, generating a novel or a conventional variant termed sqADAR2a and sqADAR2b, respectively. The extra dsRBD of sqADAR2a increases its editing activity in vitro. We hypothesized that the high activity is due to an increase in the affinity of the enzyme for dsRNA. This may be important because protein-RNA interactions can be influenced by physical factors. We became particularly interested in analyzing the effects of salt on interactions between sqADAR2 and RNA because squid cells have a ～3-fold higher ionic strength and proportionally more Cl(-) than vertebrate cells. To date, in vitro biochemical analyses of adenosine deamination have been conducted using vertebrate-like ionic strength buffers containing chloride as the major anion, although the vast majority of cellular anions are known to be organic. We found that squid-like salt conditions severely impair the binding affinity of conventional ADAR2s for dsRNA, leading to a decrease in nonspecific and site-specific editing activity. Inhibition of editing was mostly due to high Cl(-) levels and not to the high concentrations of K(+), Na(+), and organic anions like glutamate. Interestingly, the extra dsRBD in sqADAR2a conferred resistance to the high Cl(-) levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. Site-directed mutagenesis of squid ADAR2a showed that its increased affinity and editing activity are directly attributable to the RNA binding activity of the extra dsRBD.     

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma

Background

Clinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.

Methods

The effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit^+/+ mice and -deficient Kit^W/Kit^W-v mice. The outcome of mast cell reconstitution was further investigated.

Results

Airway eosinophilia and IL-13 production were augmented by poly IC in Kit^+/+ mice but not in Kit^W/Kit^W-v mice. When Kit^W/Kit^W-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in Kit^W/Kit^W-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B₄ and prostaglandin D₂ are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.

Conclusions

We conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B₄ or prostaglandin D₂.     

The evolutionary transition from RNA to DNA in early cells

Summary The evolution of genetic material can be divided into at least three major phases: first, genomes of nucleic acid-like molecules; secondly, genomes of RNA; and finally, double-stranded DNA genomes such as those present in all contemporary cells. Using properties of nucleic acid molecules, we attempt to explain the evolutionary transition from RNA alone as a cellular informational macromolecule prior to the evolution of cell systems based on double-stranded DNA. The idea that ribonucleic acid-based cellular genomes preceded DNA is based on the following: (1) protein synthesis can occur in the absence of DNA but not of RNA; (2) RNA molecules have some catalytic properties; (3) the ubiquity of purine and pyridine nucleotide coenzymes as well as other similar ribonucleotide cofactors in metabolic pathways; and (4) the fact that the biosynthesis of deoxyribonucleotides always proceeds via the enzymatic reduction of ribonucleotides.The RNA prior to DNA hypothesis can be further developed by understanding the selective pressures that led to the biosynthesis of deoxyribose, thymine, and proofreading DNA polymerases. Taken together these observations suggest to us that DNA was selected as an informational molecule in cells to stabilize earlier RNA-protein replicating systems. These arguments include the facts that (1) the 2-deoxy-containing phosphodiester backbone is more stable in aqueous conditions and in the presence of transition metal ions (such as Zn²⁺) than its ribo-equivalents; (2) the absence of proofreading activity in RNA polymerases leads to a higher rate of mutation in RNA genomes relative to DNA; (3) information in RNA degrades because of the tendency of cytosine to deaminate to uracil and the lack of a correcting enzyme; and (4) UV irradiation produces a larger number of photochemical changes in RNA molecules relative to double-stranded DNA. The absence of atmospheric UV attenuation during the early Earth environment (Hadean and early Archean) would have imposed an intense selection pressure favoring duplex DNA over other genetic information storage systems.If RNA preceded DNA as a reservior of cellular genetic information, then an RNA-replicating oligopeptide must have been one of the earliest protoenzymes from which RNA polymerase presumably evolved. We conclude that RNA polymerases are among the oldest classes of enzymes.     

The RIG‐I‐like receptor LGP2 inhibits Dicer‐dependent processing of long double‐stranded RNA and blocks RNA interference in mammalian cells

In vertebrates, the presence of viral RNA in the cytosol is sensed by members of the RIG‐I‐like receptor (RLR) family, which signal to induce production of type I interferons (IFN). These key antiviral cytokines act in a paracrine and autocrine manner to induce hundreds of interferon‐stimulated genes (ISGs), whose protein products restrict viral entry, replication and budding. ISGs include the RLRs themselves: RIG‐I, MDA5 and, the least‐studied family member, LGP2. In contrast, the IFN system is absent in plants and invertebrates, which defend themselves from viral intruders using RNA interference (RNAi). In RNAi, the endoribonuclease Dicer cleaves virus‐derived double‐stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that target complementary viral RNA for cleavage. Interestingly, the RNAi machinery is conserved in mammals, and we have recently demonstrated that it is able to participate in mammalian antiviral defence in conditions in which the IFN system is suppressed. In contrast, when the IFN system is active, one or more ISGs act to mask or suppress antiviral RNAi. Here, we demonstrate that LGP2 constitutes one of the ISGs that can inhibit antiviral RNAi in mammals. We show that LGP2 associates with Dicer and inhibits cleavage of dsRNA into siRNAs both in vitro and in cells. Further, we show that in differentiated cells lacking components of the IFN response, ectopic expression of LGP2 interferes with RNAi‐dependent suppression of gene expression. Conversely, genetic loss of LGP2 uncovers dsRNA‐mediated RNAi albeit less strongly than complete loss of the IFN system. Thus, the inefficiency of RNAi as a mechanism of antiviral defence in mammalian somatic cells can be in part attributed to Dicer inhibition by LGP2 induced by type I IFNs. LGP2‐mediated antagonism of dsRNA‐mediated RNAi may help ensure that viral dsRNA substrates are preserved in order to serve as targets of antiviral ISG proteins.     

A double-stranded RNA mycovirus from the plant pathogenic fungus, Fusarium solani f. sp. robiniae

Abstract Two kinds of double-stranded RNA (dsRNA), estimated to be 1.9 and 1.7 kb in size, were detected in the plant pathogenic fungus, Fusarium solani f. sp. robiniae . Isometric virus-like particles (VLPs), 30 nm in diameter, were recovered from cell extracts as a discrete band when centrifuged through a CsCl density gradient. The dsRNA molecules extracted from VLP preparations were identical in electrophoretic mobility to the dsRNAs obtained directly from cells. SDS-PAGE analysis of the VLPs revealed a single polypeptide of 38 kDa. The dsRNAs obtained directly from cells. SDS-PAGE analysis of the asexual cycle).     

Structural basis of double-stranded RNA recognition by the RIG-I like receptor MDA5

RIG-I, MDA5 and LGP2 are cytosolic pattern recognition receptors detecting single-stranded or double-stranded RNA in virally infected cells. The activation of RIG-I or MDA5 stimulates the secretion of type I interferons that play key roles in antiviral immune responses. The C-terminal domains (CTD) of RIG-I and LGP2 are responsible for RNA binding; however, it is not clear how MDA5 binds RNA. To understand the structural basis of dsRNA recognition by MDA5, we have determined the 1.45 Å resolution structure of the C-terminal domain of human MDA5. The structure revealed a highly conserved fold similar to the structures of RIG-I and LGP2 CTDs. NMR titration of MDA5 CTD with dsRNA demonstrated that a positively charged surface is involved in dsRNA binding. Mutagenesis and RNA binding studies showed that electrostatic interactions play primary roles in dsRNA recognition by MDA5. Like RIG-I and LGP2, MDA5 CTD preferentially binds dsRNA with blunt ends, but does not associate with dsRNA with either 5′ or 3′ overhangs. Molecular modeling of MDA5 CTD/dsRNA complex suggests that MDA5 CTD may recognize the first turn of blunt-ended dsRNA in a similar manner as LGP2.     

The roles of plant dsRNA-binding proteins in RNAi-like pathways

Dicers are associated with double-stranded RNA-binding proteins (dsRBPs) in animals. In the plant, Arabidopsis, there are four dicer-like (DCL) proteins and five potential dsRBPs. These DCLs act redundantly and hierarchically. However, we show there is little or no redundancy or hierarchy amongst the DRBs in their DCL interactions. DCL1 operates exclusively with DRB1 to produce micro (mi)RNAs, DCL4 operates exclusively with DRB4 to produce trans-acting (ta) siRNAs and 21nt siRNAs from viral RNA. DCL2 and DCL3 produce viral siRNAs without requiring assistance from any dsRBP. DRB2, DRB3 and DRB5 appear unnecessary for mi-, tasi-, viral si-, or heterochromatinising siRNA production but act redundantly in a developmental pathway.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

In vitro analysis of RNA interference in Drosophila melanogaster

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos. These lysates reproduce all of the known steps in the RNAi pathway in flies and mammals. Here we explain how to prepare and use Drosophila embryo lysates to dissect the mechanism of RNAi.